The PI's long-term objectives are to understand the mechanism of microbial biofilm resistance to antibiotics, and to develop effective anti-biofilm therapies. This is a collaborative project that brings together the expertise of the PI in microbial multidrug resistance and the expertise and advanced genomics tools to study P. aeruginosa that have been developed by the co-Investigator and colleagues at PathoGenesis. The mechanism of extremely high resistance of microbial biofilms to antibiotics is poorly understood. Data from the literature and the PI's own findings indicate that biofilm cells might be expressing antibiotic resistance mechanisms. Finding genes responsible for biofilm resistance to antibiotics is the main goal of this application. There are two specific aims. In Specific aim 1, the PI will identify genes specifically expressed in biofilms. Two groups of experiments are proposed. (a) In vitro studies. Biofilm RNA will be isolated, labeled and used to probe a P. aeruginosa DNA array. A detailed analysis of factors likely to affect expression of biofilm genes will be performed. These factors will include the age of the biofilm, various growth conditions, including those emulating growth in vivo, and growth in the presence of antibiotics which might induce resistance. (b) In vivo studies. Sputum samples that have been collected from patients with cystic fibrosis harboring P. aeruginosa biofilm infections will be used to isolate RNA and obtain gene expression profiles. This information will allow the PI to identify biofilm-specific genes that are expressed in vivo. In Specific aim 2, the PI proposes to validate candidate genes expressed in the biofilm. Three types of experiments are proposed. (a) Mutants from an ordered Tn insertion library will be used to test involvement in antibiotic resistance of genes whose expression is changed in biofilms. Each candidate mutant will be tested in detail with a representative panel of antibiotics. This experiment will show which specific biofilm genes are responsible for increased resistance. In a complementary approach, a complete Tn insertion library will be screened for mutants with increased antibiotic susceptibility. Candidate mutants from this screen will then be tested in detail with a larger panel of antibiotics at a broad range of concentrations. (b) Possible "Universal" resistance genes will be identified by comparison with genes expressed in E. coli biofilms and probed with an E. coli DNA array. (c) P. aeruginosa mutants in genes participating in antibiotic resistance will be used to obtain expression profiles. This information will likely contribute to the understanding of the mechanism of resistance. Resistance genes identified in this project will serve as targets for drug discovery and development of effective anti-biofilm therapies.